Excess oxidation is a major threat to the genomic integrity of most living organisms. Reactive oxygen species oxidize deoxynucleoside triphosphates (dNTPs), as well as DNA, and some of the oxidized dNTPs have been shown to be mutagenic when they are incorporated in DNA. 8-Oxo-7,8-dihydro-2′-deoxyguaniosine 5′-triphosphate (8-OH-dGTP) leads to A · T-to-C · G transversions when it is incorporated opposite adenine (A) in the template (5, 14). To counteract the mutagenic 8-OH-dGTP, Escherichia coli has a sanitizing enzyme, MutT, that hydrolyzes 8-OH-dGTP (20). When the mutT gene is inactivated, the frequency of mutation of A · T to C · G increases more than a thousandfold compared with the wild-type frequency (35). In the case of 2-oxo-1,2-dihydro-2′-deoxyadenosine 5′-triphosphate (2-OH-dATP), G · C-to-T · A transversions occur when it is incorporated opposite guanine (G) in the template (14, 16). Another sanitizing enzyme, Orf135, degrades 2-OH-dATP in E. coli, and G · C-to-T · A mutations occur in an orf135-deficient strain more frequently than in the wild-type strain (15, 17).

The members of the Y family of DNA polymerases (DNA Pols) are involved in error-free and error-prone translesion synthesis (TLS) of damaged template DNA in various species, including humans (13, 26). Recently, involvement of Y-family DNA polymerases in the incorporation of damaged dNTPs was suggested by in vitro experiments performed with purified DNA Pols (28). The archaeal Y-family DNA Pols from Sulfolobus sp. and the human DNA Pols exclusively incorporate 8-OH-dGTP opposite A in the template DNA and incorporate 2-OH-dATP opposite G and thymine (T). Thus, it would be interesting to examine the in vivo roles of Y-family DNA Pols in the incorporation of mutagenic dNTPs into DNA. Escherichia coli strain QC1736 seems to be an appropriate background to investigate the roles of Y-family DNA Pols (DNA Pol IV and Pol V encoded by dinB and umuDC, respectively) in the mutagenesis caused by oxidized nucleotides. Iron metabolism is deregulated in this strain due to the lack of the Fur protein, a negative regulator of iron uptake (29). This strain also lacks both superoxide dismutases (SodA and SodB), which catalyze the breakdown of the superoxide anion. Thus, both iron overload and superoxide stress occur in strain QC1736, which leads to high rates of spontaneous mutation from A · T to C · G and from G · C to T · A (24). The hot spots and sequence contexts of A · T-to-C · G mutations are almost identical to those in a mutT strain (25). In contrast, the hotspots of G · C-to-T · A mutations are very different from those in mutM mutY strains, in which 8-OH-G in DNA acts as a major mutagenic lesion. Expression of the cDNA of the human counterpart of E. coli MutT, MTH1, which hydrolyzes both 8-OH-dGTP and 2-OH-dATP (12), suppresses the mutator phenotype of the strain. Thus, it has been concluded that the targets contributing to the oxidative mutagenesis in the sod/fur mutant are oxidized dNTPs, such as 8-OH-dGTP and 2-OH-dATP, rather than DNA (25).

To examine the in vivo roles of Y-family DNA Pols, mutation frequencies were compared for sod/fur strains with and without Pol IV and Pol V (Table 1). Both A · T-to-C · G and G · C-to-T · A transversion frequencies were reduced by 80 to 90% by deletion of either dinB or umuDC or both in the sod/fur strains (Table 2). Interestingly, the double mutants (ΔdinB ΔumuDC) exhibited levels of mutation frequency similar to those of single mutants (ΔdinB or ΔumuDC). These results suggest that the base substitutions by erroneous incorporation of 2-OH-dATP or 8-OH-dGTP require both DNA Pol IV and DNA Pol V functions.

The dNTP pool and DNA are continuously exposed to a variety of exogenous and endogenous damaging agents, including reactive oxygen species, and the incorporation of oxidized dNTPs into DNA is a major source of spontaneous mutagenesis and carcinogenesis (1). Here we obtained biochemical and genetic evidence that DNA Pol IV and Pol V may be involved in oxidative mutagenesis through misincorporation of altered nucleotides (i.e., 2-OH-dATP and 8-OH-dGTP) during DNA synthesis. This is consistent with the report by Satou et al. (27) that DNA Pol IV promotes mutation of G · C to T · A in E. coli when 2-OH-dATP is directly introduced into cells by CaCl2 treatment. It has also been suggested that SOS-inducible polymerases, including Pol IV and Pol V, are involved in mutagenesis caused by increases in the normal levels of dNTPs (33). It has been reported that more than one DNA polymerase is involved in mutagenesis when the Y-family DNA polymerases are involved in TLS. For benzo[a]pyrene-induced mutagenesis, both Pol IV and Pol V are required for a −1 frameshift TLS (23). DNA lesions induced by other chemicals, including 3-methylcholanthrene or dimethylbenzo[a]anthracene, also require both DNA Pol IV and Pol V for a −2 frameshift in a CG repetitive sequence in Salmonella enterica serovar Typhimurium (18, 21). Thus, we speculate that DNA Pol IV and Pol V are involved in sequential biochemical steps, such as incorporation and extension of oxidized dNTPs during chromosome replication. One of these polymerases might incorporate oxidized dNTPs into DNA in an erroneous manner, and the other might extend the mutagenic primer termini containing the oxidized deoxynucleoside monophosphate, thereby inducing base substitutions. It is obvious, however, that more experiments are needed to elucidate the precise mechanisms.

DNA Pol IV is controlled by σs, and the level of expression of Pol IV in the stationary phase decreases significantly when the rpoS gene encoding σs is defective (10, 19). Thus, Pol IV appears to be regulated not only by the SOS response but also by the σs-dependent stress response. In stationary-phase cells, the amount of cellular mismatch repair proteins decreases at least 10-fold (8). Hence, the error-prone nature of Pol IV is expected to be more significant. In fact, DNA Pol IV is responsible for some of the adaptive mutations in stationary-phase cells (9, 22), Interestingly, adaptive mutagenesis is approximately fourfold more frequent in a sodA sodB strain than in the parental strain, and this mutagenesis is suppressed under anaerobic conditions (2). Therefore, DNA Pol IV might be involved in stationary-phase mutagenesis by either incorporation of oxidized dNTPs or extension of primers having oxidized deoxynucleoside monophosphates or both, although it is possible that DNA Pol IV induces mutations by error-prone bypass across oxidized bases in template DNA.

The oxidized nucleotide pools also cause a problem in mammalian cells. Spontaneous tumorigenesis in lungs, livers, and stomachs is enhanced in mice that are deficient in Mth1 (30). In addition, a recent study suggested that the majority of mutations in human cells that are deficient in mismatch repair do not arise from spontaneous replication errors but from the incorporation of oxidized dNTPs (6). Thus, it might be interesting to examine the roles of mammalian Y-family DNA Pols in genome instability caused by oxidation of the nucleotide pool.

Incorporation of oxidized nucleotides by DNA polymerases. The incorporation of 2-OH-dATP (A) and 8-OH-dGTP (B) into DNA by DNA Pol IV and KF exo− of E. coli was assayed as described previously (28). Cy3-conjugated primer, annealed to the template at a 1:1 ratio (0.1 μM), was incubated with DNA Pol IV (0.1 μM) or KF exo− (0.02 U), and then 50 μM 2-OH-dATP (A) or 50 μM 8-OH-dGTP (B) was added. No other dNTPs were added to the reaction mixtures. All the reactions were carried out at room temperature for 30 min. The reaction products were analyzed on 15% denatured polyacrylamide gels, and the bands were visualized using a Molecular Imager FX Pro system (Bio-Rad, Richmond, CA). The oligonucleotide sequences of the primer and template were 5′-Cy3-CGCGCGAAGACCGGTTAC-3′ and 5′-GAAGGGATCCTTAAGACNGTAACCGGTCTTCGCGCG-3′, respectively, for 2-OH-dATP and 5′-Cy3-CGGAGCTCGGTCGGCGTCTGCGTC and 5′-AGCCGCAGGAGNGACGCAGACGCCGACCGAGCTCCG-3′, respectively, for 8-OH-dGTP (N = A, C, G, or T). Parts of the sequences of the primer and template are shown. The unlabeled lanes on the left indicate the positions of Cy3-labeled primers without extension.

ACKNOWLEDGMENTS

Part of this study was financially supported by the Budget for Nuclear Research of the Ministry of Education, Culture, Sports, Science and Technology Japan. This work was also supported by grant-in-aid for international collaborative research SH34407from the Japan Health Science Foundation.